Background: Goal-directed ultrasound protocols have been developed to facilitate efficiency, throughput, and patient care. Hands-on instruction and training workshops have been shown to positively impact ultrasound training. Objectives: We describe a novel undifferentiated chest pain goal-directed ultrasound algorithm-focused education workshop for the purpose of enhancing emergency medicine resident training in ultrasound milestones competencies. Methods: This was a cross-sectional study performed at an academic medical center. A novel goal-directed ultrasound algorithm was developed and implemented as a model for teaching and learning the sonographic approach to a patient with undifferentiated chest pain. This algorithm was incorporated into all components of the 1-day workshop: asynchronous learning, didactic lecture, case-based learning, and hands-on stations. Performance comparisons were made between postgraduate year (PGY) levels. Results: A total of 38 of the 40 (95%) residents who attended the event participated in the chest pain objective standardized clinical exam, and 26 of the 40 (65%) completed the entire questionnaire. The average number of ultrasounds performed by resident class year at the time of our study was as follows: 19 (standard deviation [SD]=19) PGY-1, 238 (SD=37) PGY-2, and 289 (SD=73) PGY-3. Performance on the knowledge-based questions improved between PGY-1 and PGY-3. The application of the novel algorithm was noted to be more prevalent among the PGY-1 class. Conclusion: The 1-day algorithm-based ultrasound educational workshop was an engaging learning technique at our institution.

In the following work, nanoparticle quantum dot (QD) fluorophores have been exploited to measure biologically relevant analytes via a miniaturized sensor ensemble to provide key diagnostic and prognostic information in a rapid, yet sensitive manner—data essential for effective treatment of many diseases including HIV/AIDS and cancer. At the heart of this “nano-bio-chip” (NBC) sensor is a modular chemical/cellular processing unit consisting of either a polycarbonate membrane filter for cell-based assays, or an agarose bead array for detection of biomarkers in serum or saliva. Two applications of the NBC sensor system are described herein, both exhibiting excellent correlation to reference methods ((R² above 0.94), with analysis times under 30 minutes and sample volumes below 50 [mu]L. First, the NBC sensor was employed for the sequestration and enumeration of T lymphocytes, cells specifically targeted by HIV, from whole blood samples. Several different conjugation methods linking QDs to recognition biomolecules were extensively characterized by biological and optical methods, with a thiol-linked secondary antibody labeling scheme yielding intense, specific signal. Using this technique, the photostability of QDs was exploited, as was the ability to simultaneously visualize different color QDs via a single light pathway, effectively reducing optical requirements by half. Further, T-cell counts were observed well below the 200/[mu]L discriminator between HIV and AIDS and across the common testing region, demonstrating the first reported example of cell counting via QDs in an enclosed, disposable device. Next, multiplexed bead-based detection of cancer protein biomarkers CEA, Her-2/Neu, and CA125 in serum and saliva was examined using a sandwich immunoassay with detecting antibodies covalently bound to QDs. This nano-based signal was amplified 30 times versus molecular fluorophores and cross talk in multiplexed experiments was less than 5%. In addition, molecular-level tuning of recognition elements (size, concentration) and agarose porosity resulted in NBC limits of detection two orders of magnitude lower than ELISA, values competitive with the most sensitive methods yet reported (0.021 ng/mL CEA). Taken together, these efforts serve to establish the valuable role of QDs in miniaturized diagnostic devices with potential for delivering biomedical information rapidly, reliably, and robustly. / text

A magnetic bead-based DNA extraction and purification device has been designed to be used for extraction of the target DNA molecules from whole blood sample. Mixing and separation steps are performed using functionalised superparamagnetic beads suspended in the cell lysis buffer in a circular chamber that is sandwiched between two electromagnets. Non-uniform nature of the magnetic field causes temporal and spatial distribution of the beads within the chamber. This process efficiently mixes the lysis buffer and whole blood in order to extract DNA from target cells. Functionalized surface of the magnetic beads then attract the exposed DNA molecules. Finally, DNA-attached magnetic beads are attracted to the bottom of the chamber by activating the bottom electrode. DNA molecules are extracted from the magnetic beads by washing and re-suspension processes. The numerical simulation approach has been adopted in order to design the magnetic field source. The performance of the magnetic field source has been investigated against different physical and geometrical parameters and optimised dimensions are obtained with two different magnetic field sources; integrated internal source and external source. A new magnetic field pattern has been introduced in order to efficiently control the bulk of magnetic beads inside the mixing chamber by dynamic shifting of magnetic field regions from the centre of the coils to the outer edge of the coils and vice versa. A Matlab code has been developed to simulate beads trajectories inside the designed extraction chip in order to investigate the efficiency of the magnetic mixing. A preliminary target molecule capturing simulation has also been performed using the simulated bead trajectories to evaluate the DNA-capturing efficiency of the designed extraction chip. The performance of the designed extraction chip has been tested by conducting a series of biological experiments. Different magnetic bead-based extraction kits have been used in a series of preliminary experiments in order to extract a more automation friendly extraction protocol. The efficiency of the designed device has been evaluated using the spiked bacterial DNA and non-pathogenic bacterial cell cultures (B. subtilis, Gram positive bacteria and E. coli, Gram negative bacteria) into the blood sample. Excellent DNA yields and recovery rates are obtained with the designed extraction chip through a simple and fast extraction protocol.

This dissertation discusses the development of inexpensive, easy-to-use, and field-deployable diagnostic techniques and devices for the early detection of various pathogens, commonly found in clinical samples and contaminated food and water. Infectious diseases account for about 90% of world health problems, killing approximately 14 million people annually, the majority of which reside in developing countries. In 2012, the World Health Organization (WHO) published data on the top 10 causes of death across the globe. Although communicable disease is a prevalent cause of fatality, both low-income and high-income countries, pathogen species and transmission are very different. Nearly 60% of deaths in developing countries are caused by food, water, air or blood-borne pathogens. The most prevalent illnesses are diarrheal disease, malaria, and HIV/AIDS. By contrast, the leading causes of death in developed countries (heart disease, cancer, and stroke) are not communicable and are often preventable. However, there is an increasing need for the development of rapid and accurate methods for pathogen identification in clinical samples, due to the growing prevalence of antibiotic-resistant strains. Incorrect, or unneeded antibiotic therapies result in the evolution of extremely aggressive nosocomial (hospital-acquired) infections, such as methicillin- (MRSA) and vancomycin-resistant Staphylococcus aureus (VRSA). The implementation of rapid, easy to use and cost-effective diagnostics will reduce the frequency of pathogen-related deaths in underdeveloped countries, and improve targeted antibiotic treatment in hospital settings, thus decreasing the potential development of more treatment-resistant "super bugs". This research includes novel techniques utilizing two major sensing modalities: serological (i.e. immunological), and nucleic acid amplification testing (NAATs). We first developed a highly sensitive (limit-of-detection = 100 CFU mL-1) particle immunoassay that takes advantage of elastic and inelastic light scatter phenomena, for optical detection of target antigens. This assay is performed upon a unique nanofibrous substrate that promotes multiplexing on a user-friendly platform. We then developed a novel technique, termed emulsion loop-mediated isothermal amplification (eLAMP), in which the target amplicon is detected in real-time, again utilizing light scattering detection and quantification. Both techniques require no sample pre-treatments, and can be combined with smartphone imaging for detection of targets in under 15 minutes. These methods have the potential to improve the speed and sensitivity of early pathogenic identification, thus leading to a reduction in preventative deaths and a decrease in global economic costs associated with infectious disease in clinical and other settings.

The South African National Antiretroviral Treatment Guidelines recommend the use of HIV-1 viral load assays for routine monitoring of HIV-1 positive patients receiving highly active antiretroviral therapy (HAART). This thesis describes the innovative approaches to developing more affordable HIV-1 diagnostics and monitoring assays for South Africa, which take into account the tiered laboratory infrastructure of this country. An in-house HIV-1 viral load assay – the LUX assay, was developed and evaluated with a view of implementing this more affordable option in high tier laboratories. The LUX assay represents quantitative real-time RT-PCR that utilizes the LightCycler® technology (Roche) in a novel combination with a LUXÔ primer. The assay showed good analytical sensitivity, specificity and reproducibility of its linear dynamic range of 4x102 to 4x106 RNA copies/ml. Preliminary clinical evaluation (n = 458) of the LUX assay showed good agreement with the COBAS Amplicor assay, and demonstrated its usefulness for long term monitoring of HAART patients. ELISA based viral load testing approaches were investigated as low cost and less technically complex alternatives for medium tier laboratories. The HiSens HIV-1 p24 Ag Ultra (Perkin Elmer) and the ExaVir™ Load Quantitative HIV-RT kits (CAVIDI) were compared with the Roche Amplicor assay. Both assays showed strong association with the Roche Amplicor assay, with R2 = 0.686 and R2 = 0.810, respectively (n = 117). These alternative assays seemed most useful in the serial monitoring of patients on HAART. Major drawbacks included the wide variability of both assays, insufficient sensitivity of the p24 antigen assay and low throughput of the RT assay. Development of a point-of-care HIV-1 RNA assay could address issues related to early and cost effective diagnosis of acute HIV infection. A novel isothermal amplification technique termed the Reverse Transcription Loop Dependant Amplification (RT-LDA) was developed as one component for a potential point-of-care HIV-1 RNA assay. The RT-LDA converted RNA into partially looped ssDNA amplicons, over a wide RNA concentration range (4x103 to 4x108 copies/ml) using a 1 hour incubation at 53ºC. The RT-LDA technology is fully compatible with a lateral flow detection system using dipsticks and highly suitable for point-of-care testing. Overall, this study demonstrates the feasibility of developing novel, more affordable HIV-1 testing options that would be appropriate for the tiered laboratory infrastructure present in South Africa. Evaluation of commercially available, less expensive alternative HIV viral load assays in local settings facilitates their implementation.

Clubfoot is a congenital foot disorder that, left untreated, can limit a person's mobility by making it difficult and painful to walk. Although inexpensive and reliable treatment exists, clubfoot often goes untreated in the developing world, where 80% of cases occur. Many nonprofit and non-governmental organizations are partnering with hospitals and clinics in the developing world to provide treatment for patients with clubfoot, and to train medical personnel in the use of these treatment methods. As a component of these partnerships, clinics and hospitals are collecting patient records. Some of this patient information, such as photographs, requires expert quality assessment. Such assessment may occur at a later date by a staff member in the hospital, or it may occur in a completely different location through the web interface. Photographs capture the state of a patient at a specific point in time. If a photograph is not taken correctly, and as a result, has no clinical utility, the photograph cannot be recreated because that moment in time has passed. These observations have motivated the desire to perform real-time classification of clubfoot images as they are being captured in a possibly remote and challenging environment. In the short term, successful classification could provide immediate feedback to those taking patient photos, helping to ensure that the image is of good quality and the foot is oriented correctly at the time of image capture. In the long term, this classification could be the basis for automated image analysis that could reduce the workload of a busy staff, and enable broader provision of treatment.

<p>The superior sensitivity and selectivity obtained with mass spectrometry (MS) is hardly matched by other analytical technologies, therefore it is an indispensable tool for modern society. Traditionally, MS is coupled with chromatography separation and performed in centralized analytical laboratories, which often requires extensive sample preparation and expensive instrumentation. With the advancements in the field of ambient MS and miniature MS, MS analysis at point-of-care (POC) has become a reality. Ambient MS includes a variety of methods for sampling and ionization, but they all share a common feature: they require little to no sample preparation. This has made rapid analysis of untreated sample possible and speed of MS analysis is significantly improved. Miniature MS, on the other hand, shrinks down the sizes of conventional benchtop instruments so they become portable or fieldable. In this dissertation, I documented the developments of ambient MS methods and applications of miniature MS for a variety of health-related topics, which include preclinical pharmacokinetics, intraoperative diagnosis, drug adherence monitoring and food safety. </p>

Methicillin Resistant Staphylococcus aureus (MRSA) is one of the most important threats of nosocomial infections in many regions of the world and Vancomycin Resistant Enterococcus (VRE) is an emerging pathogen that develops full resistance against third-generation glycopeptide antibiotics. Conventional methods for identification of MRSA and VRE generally depend on culturing, which requires incubation of biological samples at least 24-72 hours to get accurate results. These methods are time consuming and necessitate optical devices and experts for evaluation of the results. On the other hand, early diagnosis and initiation of appropriate treatment are necessary to decrease morbidity and mortality rates. Thus, new diagnostic systems are essential for rapid and accurate detection of biological analytes at the point of care. This study presents design, fabrication, and implementation of MEMS based micro electrochemical sensor (&micro / ECS) to detect the methicillin resistance in Staphylococcus aureus and vancomycin resistance in Enterococcus species. To the best of our knowledge, the developed sensor is the first &micro / ECS which utilizes on-chip reference (Ag), working (Au), and counter (Pt) electrodes together with a microchannel to detect MRSA and VRE. The characterization of the designed sensor was achieved analyzing the interactions of the buffer solutions and solvents with the electrodes and Parylene C film layer by using optical and electrochemical methods. Specific parts of genes that are indicators of antimicrobial resistances were used in order to detect the resistances with high selectivity and sensitivity. Thus, synthetic DNA and bacterial PCR product were used as target probes in redox marker based detection and enzyme based detection, respectively. In order to enhance the hybridization, folding structures of the capture probe were investigated by using mfold Web Server. In redox marker based detection, the hybridization of DNA was indirectly detected by using Hoechst 33258 as redox marker with differential pulse voltammetry. The cross reactivity of the tests were performed by using different target probes of femA genes of S. aureus and S. epidermis, which are the major genes detected in methicillin detection assays. Consequently, amplification of signal by using horseradish peroxidase and TMB/H2O2 as substrate was achieved in order to enhance detection sensitivity. The sensor could detect 0.01 nM 23-mer specific part of mecA gene with redox marker based detection and 10 times diluted PCR product with enzyme-based detection in about six hours including the steps of sample preparation from whole blood. This sensor with its compatibility to MEMS fabrication processes and IC technology has a promising potential for a hand-held device for POC through the integration of micropotentiostat.

Point-of-care testing (POCT) devices have received increasing attention because of their potential to address the urgent need for quick and accurate diagnostic tools, especially in areas of personal care and clinical medicine. They offer several benefits over current diagnostic systems, including rapid diagnostic results in comparison to microbial cultures, simple interpretation of results, portability, and requiring no specialised laboratory equipment or technical training to operate. These are essential for diagnosing critical illnesses, such as sepsis, in areas of poor healthcare infrastructure. Sepsis, an innate physiological response to infection, is a growing problem worldwide with high associated costs and mortality rates, and affects a wide range of patients including neonates, infants, the elderly, and immunocompromised individuals. A literature review of the biomarkers of sepsis and the currently available diagnostic systems indicates the need for a biosensor capable of meeting the requirements of designing POCT systems and achieving detection of low concentrations of biomarkers. To meet these demands, two significant contributions to developing POCT platforms have been achieved and described in this thesis, including: 1) development of a colorimetric, magnetically separable biosensor that can be easily fabricated and demonstrates an easily identifiable colour response upon analyte detection, as well as the ability to capture and detection target biomarkers at low concentrations from complex solutions; and 2) tuning of the biosensor’s colorimetric response to achieve low detection limits, as well as demonstration of the versatility of the biosensor for sensing different target analytes. The developed biosensor in this work combines colour responsive polydiacetylenes and superparamagnetic iron oxide for the first time to achieve a biosensor capable of meeting these demands. The sensors exhibit identifiable colour responses to biomolecule detection, capture of a target analyte from complex solutions, sensing of different target analytes, a lower detection limit of 0.01 mg/mL, and rapid separation from solution with a common magnet. This work has been a significant demonstration of the capabilities of this biosensor as a new platform for POCT systems to diagnosis sepsis, and potentially other sensing applications.

Utilizing self-assembled monolayer nanotechnology, micro-electro-mechanical systems (MEMS) and IC technologies, a novel flexural plate-wave (FPW) biosensor is developed in this dissertation for detecting the immunoglobulin-E (IgE) concentration of human serum. The acoustic waves of the proposed FPW devices are launched by the 25-pair inter-digital transducer (IDT) input electrodes and propagated through the 4.82 £gm-thick Si/SiO2/Si3N4/Cr/Au/ZnO floating thin-plate. Since the thickness of such floating thin-plate is much smaller than the designed wavelength of FPW device (80 £gm), most of the propagating wave energy will not be dissipated into outside of thin-plate and the mass sensitivity is very high. To further reduce the insertion loss of the proposed FPW devices, two 3 £gm-thick Al reflection grating electrodes (RGE) are designed beside the input and output IDTs. To implement a FPW-based IgE biosensor, a Cr/Au electrode layer has to be deposited on the backside of the floating thin-plate to serve as a substrate for further coating the cystamine SAM/glutaraldehyde/IgE antibody layers. Once the IgE antigens of human serum are bound to the IgE antibody layer, the small change in the mass of floating thin-plate will result in a shift of center frequency of the testing FPW-based biosensor. Compared to the reference FPW biosensors, the shift of center frequency generated by the testing FPW biosensor under different IgE antigen concentration can be detected by commercial network analyzer or the frequency-shift readout system developed by our collaboration laboratory (VLSI Design Lab. of NSYSU). Compared to commercial enzyme linked immunosorbent assay (ELISA) analyzer (sample volume >25 £gl/well, testing time >60 min, dimension>40 cm ¡Ñ30 cm¡Ñ10 cm), the implemented FPW-based IgE biosensor presents a smaller sample volume (<5 £gl), faster response (<10 min) and smaller size (<9 mm¡Ñ6 mm¡Ñ0.5 mm). In addition, a very low insertion loss (-9.2 dB), a very high mass sensitivity (-6.08¡Ñ109 cm2 g-1) and a very high sensing linearity (99.46 %) of the proposed IgE biosensor can be demonstrated at 6.6 MHz center frequency. This study successfully developed a novel FPW-based allergy biosensor by MEMS technology, which has great potential to be further applied into point-of-care testing (POCT) microsystem.